September 24, 2021

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Immunotherapy: What are the challenges of CAR-T cell therapy?

Immunotherapy: What are the challenges of CAR-T cell therapy?



Immunotherapy: What are the challenges of CAR-T cell therapy?

Chemotherapy, radiotherapy, and surgery are the most traditional cancer treatments, but they are less effective and have serious side effects. Therefore, in the past decade, researchers have developed new strategies to achieve complete remission of the disease. Currently, immunotherapy has become a revolutionary cancer treatment method. There are several types of immunotherapy used to treat cancer, including adoptive cell therapy (ACT).

Chimeric Antigen Receptor (CAR) T cell therapy is a type of ACT. Autologous T cells express CAR through genetic engineering to specifically kill tumor cells. CAR-T cell therapy is an opportunity to treat patients who do not respond to other first-line cancer treatments, and has shown superior anti-tumor effects in the treatment of hematological malignancies. However, this type of therapy as a first-line clinical treatment still has many challenges to overcome.

From the perspective of pharmacy, this emerging technology is still classified as an advanced therapy. Therefore, to apply this technology, certain requirements of medical supervision must first be met. Therefore, it is necessary to analyze the elements and challenges of CAR-T cell technology, take basic, clinical, and practical factors into consideration, and adopt coping strategies, so that CAR-T technology can become an affordable treatment model.


A brief history of CAR-T cell development

Looking back at the development history of CAR-T, we must first mention the first bone marrow transplantation of leukemia patients reported by Thomas and his colleagues in 1957 and the subsequent discovery of the origin of T cells by Miller and others. However, it was not until 1986 that Steven Rosenberg reported a study on tumor infiltrating lymphocytes (TIL), which made people focus on the idea that “patients’ own immune cells can fight their own cancer.”

In 1992, Sadelain et al. successfully established a retrovirus-mediated gene transfer method to T lymphocytes, making genetic modification a means to control immunity in an experimental or therapeutic environment. Almost at the same time, Zelig Eshhar and colleagues used the antibody binding domain and the γ or ζ subunits of immunoglobulins on T cell receptors to design the specific activation of cytotoxic lymphocytes through a chimeric single chain, thereby developing the first Generation CAR-T cells.

Five years later, Dr. Sadelain’s research team proved that the integration of costimulatory signals such as CD28 into CAR-T can enhance survival, proliferation and maintain activity, thereby developing the second generation of CAR. Subsequently, CAR-T cells carrying CD19 targeting CD19 were developed, and phase I clinical trials for chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) were initiated. The test results proved that CAR-T therapy induces effective remission in adults with chemotherapy-refractory ALL, and the scale of bioprocess production was subsequently expanded.

In 2017, the FDA approved CD19 CAR-T cell therapy (Tisagenlecleucel) for ALL in children and young adults. So far, the FDA has approved five CAR-T cell therapy drugs for cancer treatment.

Immunotherapy: What are the challenges of CAR-T cell therapy?


The clinical challenges of CAR-T cell therapy

The challenges faced by CAR-T cell therapy are mainly related to side effects, toxicity, T cell exhaustion and malignant tumor microenvironment (TME). In addition, the manufacturing process in large-scale production is currently time-consuming and expensive. Therefore, it is a greater challenge to make as many patients as possible receive CAR-T cell immunotherapy.

Immunotherapy: What are the challenges of CAR-T cell therapy?

Side effects and toxicity

After CAR-T cell infusion, this immunotherapy may have potentially fatal toxicity. According to reports, some side effects include fever, inflammation, abnormally elevated liver enzymes, difficulty breathing, chills, confusion, dizziness, severe nausea, vomiting and diarrhea. All patients have long-term B cell aplasia, which can be relieved by taking gamma globulin. There are two main types of toxicity: cytokine release syndrome (CRS) and neurotoxicity (NTX) or CAR-T cell-related encephalopathy syndrome (CRES).

CRS or “cytokine storm” is a systemic inflammatory response that is caused by a large number of activated lymphocytes (B cells, T cells, and natural killer cells) and myeloid cells (macrophages, dendritic cells, and monocytes). The clinical symptoms include fever, fatigue, headache, skin rash, arthralgia and myalgia. CRS is the most common adverse reaction that occurs within a few days after the first CAR-T cell infusion (85% of patients have any grade of CRS). Severe CRS cases are characterized by tachycardia, hypotension, pulmonary edema, cardiac insufficiency, high fever, hypoxia, kidney damage, liver failure, coagulopathy, and irreversible organ damage. Fortunately, the effects of CRS can be alleviated by reducing the number of infused T cells and/or by taking anti-IL-6 receptor monoclonal antibodies and steroids.

NTX is another common complication of CAR-T cell immunotherapy, occurring in more than 40% of patients. It usually occurs within 1 to 3 weeks after CAR-T cell infusion, and is usually associated with CRS. The patient showed various symptoms, such as confusion, dullness, tremor, delirium, difficulty finding words, and headache; other symptoms such as aphasia, cranial nerve abnormalities, and epilepsy have also been reported.

Timely management of toxicity is essential to reduce mortality associated with immunotherapy. Therefore, researchers have developed different safety strategies to overcome and prevent CAR-T cytotoxicity, such as designing a new generation of CAR. Toxicity management has become a key step for the success of CAR-T cell immunotherapy.

CAR-T cell depletion

Although CAR-T cell therapy has a high complete remission rate, most patients who have achieved remission show disease recurrence within a few years. The recurrence rate of B-ALL ranges from 21% to 45%, and it increases with the extension of follow-up time. . Part of the reason for treatment failure is the depletion of CAR-T cells caused by TME produced by solid tumors.

CAR-T cell exhaustion refers to a state of dysfunction, which is characterized by the loss of antigen-specific T cells due to continuous antigen stimulation, increased expression of the costimulatory domain of the CAR structure and inhibitory receptors. In vitro CAR-T cell studies have shown that in the process of CAR-T cell depletion, the expression of inhibitory receptors (such as PD-1, Lag3, Tim3 and TIGIT) is up-regulated, and the PI3K/AKT pathway is inhibited by CTLA-4. The main reason for the loss of anti-tumor function. Cytokines also play an important role in this, such as exhausted CAR-T cells reduce the ability to express and secrete IL-2, TNF-α and IFN-γ. Other factors, such as transcription factors, metabolism, and epigenetic modifications, also play an important role in the development of CAR-T cell depletion.

One possible way to delay depletion is to construct CAR-T cells that are resistant to depletion. Recent reports indicate that the discovery of certain transcription factors such as TOX and NR4A, as well as the lack or overexpression of the AP-1 family transcription factor c-Jun, increase the resistance of CAR-T cells to exhaustion. Recently, the knockout of PD-1 through CAR-T cell engineering (CRISPR/Cas9) or the use of PD-1 blocking antibodies have been used to improve the therapeutic effect of CAR-T and avoid depletion.

Tumor microenvironment

CAR T cell immunotherapy has not been successful in solid tumors. One possible reason is that the immunosuppressive properties of TME affect the efficacy of adoptive immunotherapy. Solid tumors have highly infiltrating mesenchymal cells, such as cancer-associated fibroblasts (CAF) and suppressive immune cells, including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAM), and tumor-associated neutrophils ( TAN), mast cells, and regulatory T cells (Treg), which help establish an immunosuppressive TME that can interfere with the efficacy of CAR-T cell therapy.

Strategies to overcome the effect of TME include enabling T cells to resist tumor suppression in TME, such as the transgene expression of dominant negative receptors or signal converters, which can convert inhibitory signals into stimulus signals. Another opportunity to overcome the persistence and depletion of CAR-T cells is to improve drug delivery to the tumor site. For CAR-T cells, local injection is an ongoing attempt.

Genetic alteration

Some reports show that patients treated with CAR-T cells lack efficacy and relapse and have genetic changes. Orlando et al. integrated whole-exome DNA-seq and RNA-seq to study the extent of recurrence caused by CD19 mutations. They found new genetic changes in exons 2–5 of the CD19 gene in 12 patient samples, and 8 of 9 patients showed loss of heterozygosity. They concluded that CD19 homozygous mutations are acquired anti-CAR- The main reason for T cell therapy.

Asnani et al. reported similar findings. They described the skipping of exon 2 and exon 5-6 in patients with relapsed leukemia after CAR-T cell treatment. Exon 2 is essential for the integrity of the CAR-T CD19 epitope, while exons 5-6 are responsible for the CD19 transmembrane domain. However, further research is needed to explore the impact of genome analysis.


The manufacturing process challenge of CAR-T cells

The traditional techniques for manufacturing CAR-T cells include: 1) Isolate patient’s T cells (autologous) by apheresis; 2) transport the recovered cells to a central production base; 3) genetically modify them to express CAR; 4) Expansion in the laboratory; 5) Return the CAR-T cells to the hospital and inject them into the patient.

Immunotherapy: What are the challenges of CAR-T cell therapy?

The logistics involved in this traditional manufacturing and treatment using autologous CAR-T cells has increased the complexity of clinicians and patients. Today, this therapy brings some major manufacturing challenges, including:

CAR-T cell packaging, transportation and storage

The clinical manufacturing of CAR-T cells is currently a complex process involving multiple steps, spanning different geographic locations, and using multiple technologies and logistics. Any errors in time, transportation methods, cold chain, or storage can cause cell damage and directly affect the efficiency of treatment. Therefore, each step requires careful management, accurate sample tracking, and sufficient preservation technology to freeze patient samples. In the entire CAR-T cell manufacturing process, different transportation at different temperatures is required. Therefore, the cryopreservation during the production process must ensure quality control.

Good Manufacturing Practice (GMP)

CAR-T cells are a complex preparation process, and cGMP is the key and bottleneck in the production of CAR-T cells. The purpose of cGMP is to provide a framework to ensure that well-trained and regularly trained employees conduct high-quality production in well-controlled facilities and equipment. Likewise, it provides a rigorous document process covering all aspects of operations to demonstrate continuous and adequate compliance.

According to the International Organization for Standardization (ISO), CAR-T cell manufacturing requires GMP facilities as cell processing clean rooms, which must be equipped with 1) facility systems (such as air handlers, 24/7 alarm monitoring systems); 2) environmental monitoring equipment (such as particles) Counter); 3) Manufacturing process equipment (such as cell washing machine, bioreactor); 4) Analysis equipment (such as automatic cell counter, flow cytometer).

Another key factor in maintaining a GMP-compliant production environment is highly skilled employees, who should have extensive knowledge of GMP production, quality control and quality assurance.

Preparation of Lentiviral Vector (LV)

The production of LVs faces many challenges, such as its inherent cytotoxicity, low stability, and dependence on transient transfection effects. In addition, the upstream and downstream processes have low yields and low cost-effectiveness. Part of this part of the successful commercial products is the establishment of standardized and stable cell lines to produce LVs that promote the GMP compliance process, which can provide easier scale-up, reproducibility, biosafety and cost-effectiveness.

Staff and training

Taking into account the complexity of the treatment and its associated high-risk side effects, the use of CAR-T cells is highly regulated and can only be used in certification centers and managed by well-trained staff. All employees involved in CAR-T cell manufacturing (from T cell collection to manufacturer to clinical unit) need to undergo extensive training and have a satisfactory level of ability. This ability can manage the complications that may arise in the process, so that the product can be delivered.

Today, there are only a few qualified professionals in this field, and multidisciplinary collaboration and exchanges are needed to create more knowledge in this field. Academic participation is also an important aspect.

QC

As a living “drug”, CAR-T cells have a complicated preparation process and require “whole process quality control”. In the production process, well-controlled cold chain transportation and storage play an important role in ensuring the quality of cell products and preventing bacteria and mycoplasma contamination. The requirements of CAR-T cell quality control include checking whether the T cells transduced in vitro have virus replication and residual production materials.

In addition, considering the characteristics of CAR-T cells as biological products, cell products and gene therapy products, the release test of finished products should also be included to confirm their characteristics, purity, safety and efficacy.

Immunotherapy: What are the challenges of CAR-T cell therapy?

In addition, stability studies are required to verify storage conditions and their shelf life. The generation of CAR-T cells requires more in-depth research to assess the quality of T cells in patients with relapse and reinfusion. These studies should provide data on the distribution of lymphocyte populations. In short, quality control is critical to the success of CAR-T treatment.

Production scale-up

The manufacture of CAR-T cells should be scalable (ie, equip each patient with multiple single bioreactors) in order to expand the scope of benefit to patients without sacrificing product quality and repeatability. Personalized therapy (such as autologous cell therapy) does not just increase the volume like ordinary biopharmaceuticals, but requires more refined amplification, that is, having multiple bioreactors to amplify the CAR-T cells of each patient. In addition, it depends on the ability to implement multiple independent products in parallel.

Manufacturing time and repeated dosing

The production of CAR-T cells may take up to 4 weeks. During this time, patients are extremely vulnerable to the risk of disease progression and death. In addition, the manufacture of CAR-T cells does not allow for volume enlargement. Therefore, the cells must be prepared as a single batch, which limits the number of available products. In this case, patients may not have the opportunity to receive new CAR-T cell infusions quickly and conveniently.

Pricing and availability

Pricing and patient accessibility are the most important limitations for the popularization of CAR-T cell use worldwide. The current CAR-T cell manufacturing model is highly concentrated, and the process of each step is very complicated, resulting in a cost of as much as US$373,000 to US$475,000 per treatment (the hospital expenses related to the treatment are not included in this type of average cost) , Patients and the medical system can’t afford it.

This prohibitive cost limits patients’ access to treatment, especially in countries with underdeveloped socio-economic conditions, which further limits the wide application of CAR-T cell therapy. Until CAR-T cell therapy becomes economical and affordable, its therapeutic potential will not be truly realized.

Regulatory requirements

Another important bottleneck for cell products is regulation. CAR-T cells are considered Advanced Therapeutic Drug Products (ATMP) worldwide, and these products require a license. Regulatory agencies are highly related to standard therapies, but cell products have special requirements. The US or EU regulatory agencies are working hard to define the best guidelines to coordinate ATMP clinical manufacturing requirements on a global scale. At the same time, underdeveloped countries are facing greater challenges because the clinical use of CAR-T therapy is greatly restricted, resulting in a lack of understanding of regulatory requirements by the authorities.


Strategies to enhance the use of CAR-T cell technology

Discovery of new biomarkers

Biomarkers are of great significance for the clinical treatment of cancer. They can be used to determine patients suitable for CAR-T treatment, prognosis, treatment response prediction, and monitor disease progression. The first biomarker for CAR-T treatment is CD19, a B cell surface protein mainly expressed on malignant B cells.

Currently, different biomarkers are being sought according to the stage of immunotherapy, including biomarkers to determine the patient’s baseline status, CAR-T cell function, CAR-T cell exhaustion, CAR-T cytotoxicity biomarkers, and cancer prognosis, Biomarkers of response and relapse. Baseline biomarkers include cytokines, such as IL-2, IL-5, IL-7, TNF-a, etc.; lactate dehydrogenase (LDH) and CD9 cells have been widely used.

For CAR-T cell function, the following biomarkers have been proposed: CD45RA, CD45RO, CD62L, CCR7, CD27, CD28, CD25, CD127, CD57 and CD137. Currently, there are no mature biomarkers that can be used to assess CAR-T cell depletion after infusion in patients. Some indirect parameters may help achieve this goal, such as the high-level expression of inhibitory receptors such as PD-1, LAG-3, and TIM-3.

Although CAR-T therapy has made significant progress, it is still necessary to continue to explore different cancer cell type-specific biomarkers to develop more specific treatment methods.

Allogeneic CAR-T cell

Currently, most CAR-T cell immunotherapy is produced using autologous T cells. This presents several disadvantages at different levels. For example, the production process may be time-consuming and complicated, leading to increased costs. In addition, due to the use of patient-derived T cells for CAR treatment, the challenges faced include weak proliferation and expansion of CAR-T cells. Limited growth and poor continuity.

One opportunity to improve these problems is to use allogeneic CAR-T cells, thereby reducing the time delay in autologous cell production. In addition, universal CAR-T cells produced from allogeneic healthy donors are easier to obtain and of higher quality. This is very important for aggressive cancer patients who need urgent treatment. This strategy will expand the number of patients who can receive the immunotherapy, making CAR-T cell therapy a ready-made treatment with low cost and easy availability, and will improve the quality characteristics of T cells.

General CAR-T cells also face some challenges and problems. For example, the immune mismatch between the donor and recipient, if the subject’s allogeneic T cells attack healthy recipient tissues, may lead to life-threatening graft-versus-host disease (GVHD), if the subject’s immune system Recognizing and responding to allogeneic T cells, these cells may be quickly eliminated by the host’s immune system. One possible solution is to eliminate GVHD by knocking out or destroying the TCR gene and/or HLA class I locus on the donor.

Compared with CAR-T cells, CAR-NK cells provide more advantages, such as reducing cytokine release syndrome and neurotoxicity in the autologous environment; the use of IPSC can provide an unlimited amount of “ready-made” NK cells, It has a rapid response to malignant cells without causing GVHDs; another advantage is that it activates multiple mechanisms of cytotoxic activity (NKG2D, KIR, CD16, NKp30, NKp44, NKp46), and still maintains its targeting of solid tumors and drug resistance The penetration capacity of the tumor microenvironment. CAR-NK preclinical studies have shown that it is effective against hematological malignant tumor targets (CD19 and CD20) and solid tumor targets, proving its potential for allogeneic therapy.

Other strategies to improve CAR-T cell therapy

Dual-targeted or tandem CARs consist of the co-expression of two separate CARs in each T cell, and they recognize two different antigens. Some dual CARs have entered clinical trials for CD19/CD20 hematological malignancies and solid tumors. The HER2/MUC1 bispecific CAR has a good effect in the in vitro test of breast cancer model. Dual CAR is a very promising method to solve antigen heterogeneity and prevent recurrence.

In addition, synthetic Notch (synNotch) receptors have been applied to CAR-T cells to improve safety. SynNotch receptor recognizes a specific tumor antigen and then releases the transcription activation domain to promote the local expression of CAR. In addition, synNotch-regulated CAR expression can prevent constitutive signaling and depletion, leaving a higher proportion of T cells in a naive/stem cell memory state.

Inhibitory chimeric antigen receptors (iCAR) contain inhibitory receptors, such as PD-1 and CTLA-4, which play a key role in attenuating or stopping the T cell response. Therefore, they are considered a safety strategy that makes T cells can distinguish between target cells and non-target cells.

In addition, in order to effectively treat solid tumors, innovative combination strategies such as vaccines, biomaterials and oncolytic viruses are promising, because they can directly enhance the function of T cells, or recruit endogenous immune cells and reshape TME.

Fully automated manufacturing process

Worldwide, the number of patients requiring CAR-T cell immunotherapy is rapidly increasing, and the industry has developed automated and closed manufacturing platforms to adapt to this situation. Examples of this work include the automation platforms of Cocoon® (Lonza) and CliniMACS Prodigy (Miltenyi Biotec), both of which allow the replication and rapid production of cells, and each step is strictly documented. In 2020, Lonza and Sheba Medical Center announced that the first patient received CD19 CAR-T cell immunotherapy manufactured using the Lonza Cocoon platform at Sheba Medical Center.

Collaboration network

Due to the high cost and high technical requirements, most patients still cannot afford CAR-T cell therapy. In order to realize the popularization of CAR-T cells, it is necessary to establish a collaborative network among different stakeholders such as academia, industry, and hospitals, so as to formulate adequate and powerful products for such products in each country where this technology is applied. legislation.

Therefore, universities should provide future professionals with knowledge and innovation so that they can study new biomarker discovery, develop stable cell lines, develop new analytical methods to verify product quality, and understand different production systems. Industries that apply GMP, quality control, and automated processes can support the establishment of more standardized, more reliable, and higher-quality products, and can also develop on-site production devices to reduce transportation and storage costs. The hospital must have adequate facilities to manage this technology, with personnel trained in the management of on-site production units, as well as medical staff and health professionals who can effectively treat patients.

Including all these activities, strong specific legislation must be formulated in this area to ensure the quality of this type of advanced treatment. In addition, supervision is an important part of this process. Nowadays, there is little information about regulatory guidelines in different countries. EMA and FDA guidelines have established a baseline for such requirements for these technologies, but the behavior and results of different stages of clinical trials will involve new considerations.


Summary

From a pharmaceutical point of view, CAR-T cells are regarded as advanced therapeutic products, and their quality must be proven by their characteristics, safety and effectiveness. Each of these three areas faces major challenges.

Both the scientific community and the industry need to continue their efforts. In the clinical aspect, they will continue to find new biomarkers, improve the development of CARs, reduce the related adverse reactions of CRS and NTX, and the application of CAR-T cells in solid tumors to improve The safety and effectiveness of the therapy.

In terms of manufacturing and technology, lower-cost production systems, production stability, and necessary quality requirements are required. These are continuing challenges. In addition, these processes must fully comply with the requirements set by international and domestic regulatory authorities.

Finally, professionals from all walks of life are required to participate in multiple disciplines at all stages. Through close cooperation between academia, industry, hospitals, and governments at the international and regional levels, it will be possible for more patients to benefit from this new technology more broadly.

(source:internet, reference only)


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